马兜铃酸对人肾小管上皮细胞损伤的体外实验研究  

Mechanism of aristolochic acid induced injury in human renal tubular epithelial cells in vitro

作　　者：王梓华[1] 黄云剑[1] 

机构地区：[1]第三军医大学新桥医院肾内科,全军肾脏病中心,重庆市肾病研究所,重庆400037

出　　处：《第三军医大学学报》2009年第23期2302-2305,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30570871);重庆市自然科学基金(CSTC2007BB5017)~~

摘　　要：目的研究马兜铃酸(aristolochic acid,AA)对人近端肾小管上皮细胞(HK-2细胞)的损伤及可能机制。方法将细胞分为4组:正常对照组与AA30、60、120μmol/L组(n=6),分别作用于HK-2细胞培养48h后,倒置相差显微镜观察细胞形态,CCK8试剂盒(Cell Counting Kit-8)检测增殖,流式细胞仪检测凋亡,Western blot分析激活型Caspase-3的表达,全自动生化检测仪测定上清液中乳酸脱氢酶(LDH)和β-N-乙酰氨基葡萄糖苷酶(NAG酶)的含量,激光共聚焦扫描荧光显微镜(laser scanning confocal microscope,LSCM)观察α-平滑肌肌动蛋白(smooth muscle actin,α-SMA)和E-钙黏连蛋白(E-cadherin)的表达,ELISA测定上清液中转化生长因子-β1(transforming growth factor-β1,TGF-β1)和Ⅲ型胶原的分泌。结果HK-2分别在30、60、120μmol/L AA作用48h后,出现增殖抑制,凋亡增加,激活型Caspase-3表达增多,LDH和NAG酶升高,均呈剂量依赖性。60μmol/L浓度的AA还表现E-cadherin表达减弱,α-SMA表达增强,TGF-β1和Ⅲ型胶原分泌明显增加(P<0.05)。结论AA可引起HK-2细胞明显的增殖抑制、凋亡和上皮-间充质转分化呈一定的浓度依赖性。Objective To investigate the possible injury mechanism of human kidney proximal tubular epithelial cell-2 （HK-2） induced by aristolochic acid （AA）. Methods Cultured HK-2 cells were divided into 4 groups： normal control, treated by AA at the concentration of 30, 60 and 120 μmol/L for 48 h respectively. The morphological changes were observed by inverted phase contract microscopy. The cell viability was measured by the Cell Counting Kit-8 （CCK8） assay. Apoptotic cells were identified by flow cytometry. Expression of active Caspase-3 was measured by Western blot analysis. Automatic biochemical analyzer was used to detect the contents of LDH and β-N-Aeetylglucosaminidase （NAG） in the supernatant. The expression of E-cadherin and a-SMA was detected with laser scanning confoeal microscope （LSCM）. Enzyme linked immunosorbent assay （ELISA） was used to measure the levels of TGF-β1 and collagen Ⅲ in the supernatant quantitatively. Results AA inhibited HK-2 cells proliferation, induced cell apoptosis and activated Caspase-3 expression, and increased the LDH and NAG levels. All of these were in a concentration-dependent manner. AA at the concentration of 60 μmol/L inhibited E-cadherin expression, increased α-SMA expression and TGF-β1 and collagen Ⅲ secretion. Conclusion AA inhibits cell proliferation, induces apoptosis and epithelialmesenchymal transition （EMT） in HK-2 cells. AA at relatively low concentration （ ≤60 μmol/L） mainly induces EMT in HK-2 cells, while, that at high concentration （≥ 120 μ mol/L） causes apoptosis and cytotoxicity.

关 键 词：马兜铃酸 肾小管上皮细胞 转分化 凋亡 

分 类 号：R392.12[医药卫生—免疫学]